Cellular regulation of messenger RNA (mRNA) is crucial for proper gene expression. One of the major pathways used to regulate eukaryotic mRNA is 5'-to-3' decay. A critical step in this pathway is the removal of the protective methyl-guanosine cap found on the 5'-end of all eukaryotic mRNA, which commits the transcript to rapid degradation. Cleavage of the cap structure is catalyzed by the conserved decapping enzyme Dcp2, in combination with protein coactivators that modulate decapping activity. Dcp2 is essential for microRNA- mediated degradation of mRNA transcripts in Drosophila, and important for degradation of long non-coding RNAs in yeast. These two classes of non-coding RNAs are important for the maintenance of cellular equilibrium in mammals and abnormal levels of micro or long non-coding RNAs are found in many human cancers. Despite the biological importance of decapping and 5'-to-3' mRNA decay, the structural details of mRNA cap cleavage by Dcp2 and its protein-protein interactions with coactivators remain poorly understood. The decapping enzyme Dcp2 appears to regulate mRNA cap removal using a combination of conformational dynamics and protein-protein interactions. Recent studies suggest that the two domains of Dcp2 form a closed, composite active site that recognizes mRNA substrate and catalyzes cap cleavage, while coactivators may accelerate decapping by promoting or stabilizing the closed, catalytically-active conformation of Dcp2. In this proposal, a diverse set f biochemical, biophysical and genetics experiments will be used to test these hypotheses by constructing a comprehensive structural model for Dcp2 activity. The catalytically-active conformation of Dcp2 will be stabilized using transition state analogs (TSAs) that mimic cap phosphate hydrolysis in the active site of Dcp2. TSAs based on oxometallate or metal fluoride additives will be identified using a combination of small angle x-ray scattering, fluorescence polarization and enzyme inhibition experiments. TSAs that promote the active conformation of Dcp2 will be structurally characterized using NMR spectroscopy and X-ray crystallography. To investigate the structural role played by coactivators of decapping, NMR spectroscopy will be used to identify contacts between the catalytic domain of Dcp2 and coactivators Dcp1 and Edc1 that might be protein-protein interaction surfaces that promote the closed, active conformation of Dcp2. NMR structural assignments, in combination with other structural data obtained from TSA studies where possible, will be used to model how coactivators perturb the conformational equilibria of Dcp2 and affect decapping activity. Mutational analyses, using in vitro decapping kinetics and in vivo yeast complementation experiments, will be used to link structure to phenotype and confirm the biological relevance of the structural model. These studies will provide a molecular level understanding of how protein-protein interactions and conformational changes in the conserved decapping enzyme Dcp2 control mRNA cap cleavage and thus help regulate transcript stability.